Collagen mimics

ABSTRACT

Novel collagen mimics are disclosed with a tripeptide unit having the formula (Xaa-Yaa-Gly) n , where one of the positions Xaa or Yaa is a bulky, non-electron withdrawing proline derivative. By substituting a proline derivative at either the Xaa or Yaa position in the native collagen helix, the stability of the helix is increased due solely to steric effects relative to prior known collagen-related triple helices. Methods are also disclosed for making the novel collagen mimics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/808,745 filed May 26, 2006. That application is incorporated hereinby reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The inventive embodiments disclosed herein were made with United Statesgovernment support awarded by the following agency: NIH AR044276. TheUnited States has certain rights in this disclosure.

BACKGROUND

Collagen is the most abundant protein in vertebrates, occurring invirtually every tissue, including skin, tendon, bone, blood vessel,cartilage, ligament, and teeth. Collagen serves as the fundamentalstructural protein for vertebrate tissues. Collagen abnormalities areassociated with a wide variety of human diseases, including arthritis,rheumatism, brittle bones, atherosclerosis, cirrhosis, and eyecataracts. Collagen is also critically important in wound healing.Increased understanding of the structure of collagen, and of how itsstructure affects its stability, facilitates the development of newtreatments for collagen-related diseases and improved wound healingtreatments.

Collagen is a fibrous protein consisting of three polypeptide chainsthat fold into a triple helix, Jenkins & Raines Nat. Prod. Rep.,19:49-59 (2002). Mammals produce at least 17 distinct polypeptide chainsthat combine to form at least 10 variants of collagen. In each of thesevariants, the polypeptide chains of collagen are composed ofapproximately 300 repeats of the tripeptide sequence Xaa-Yaa-Gly, whereXaa is often a proline (Pro) residue and Yaa is often a4(R)-hydroxyproline (Hyp) residue. In connective tissue (such as bone,tendon, cartilage, ligament, skin, blood vessels, and teeth), individualcollagen molecules are wound together in tight triple helices. Thesehelices are organized into fibrils of great tensile strength, Jones &Miller, J. Mol. Biol., 218:209-219 (1991). Varying the arrangements andcross linking of the collagen fibrils enables vertebrates to supportstress in one-dimension (tendons), two-dimensions (skin), orthree-dimensions (cartilage).

In vertebrates, the collagen polypeptide is translated with the typicalrepeat motif being ProProGly. Subsequently, in vivo, the hydroxylationof Pro residues is performed enzymatically after collagen biosynthesisbut before the chains begin to form a triple helix. Thus, hydroxylationcould be important for both collagen folding and collagen stability. Thehydroxyl group of Hyp residues has long been known to increase thethermal stability of triple-helical collagen, Berg & Prockop, Biochem.Biophys. Res. Comm., 52:115-120 (1973). For example, the meltingtemperature of a triple helix of (ProHypGly)₁₀ chains is 58° C., whilethat of a triple helix of (ProProGly)₁₀ chains is only 24° C.,Sakakibara et al., Biochem. Biophys. Acta, 303:198-202 (1973). Inaddition, the rate at which (ProHypGly)₁₀ chains fold into a triplehelix is substantially greater than the corresponding rate for(ProProGly)₁₀ chains, Chopra & Ananthanarayanan, Proc. Natl. Acad. Sci.USA, 79:7180-7184 (1982).

In general, molecular modeling based on the structure of triple-helicalcollagen and conformational energy calculations suggest that hydrogenbonds cannot form between the hydroxyl group of Hyp residues and anymain chain groups of any of the collagen molecules in the same triplehelix, Okuyama et al., J. Mol. Biol., 152:247-443 (1981). Also, severalmodels include the hypothesis that hydroxyproline increases thestability of collagen as a result of a bridge of water molecules formedbetween the hydroxyl group and a main chain carbonyl group. For reviewsof observations advancing this hypothesis, see: Suzuki et al., Int. J.Biol. Macromol., 2:54-56 (1980), and Némethy, in Collagen, published byCRC press (1988), and the references cited therein.

However, there exists experimental evidence that is inconsistent withthe bridging the water molecule model. For example, the triple helicesof (ProProGly)₁₀ and (ProHypGly)₁₀ were found to be stable in1,2-propanediol, and Hyp residues conferred added stability in theseanhydrous conditions, Engel et al., Biopolymers, 16:601-622 (1977),suggesting that water molecules do not play a part in the addedstability of (ProHypGly)₁₀. In addition, heat capacity measurements areinconsistent with collagen having more than one bound water per sixXaa-Yaa-Gly units, Hoeve & Kakivaya, J. Phys. Chem., 80:754-749 (1976).There exists no prior definitive demonstration of the mechanism by whichthe hydroxyproline residues stabilize collagen triplexes. Therefore, themolecular basis for these observed effects is still not clear. However,recent structural studies have begun to shed light on the structure andstability of collagen's triple-helix, see: Jenkins & Raines, Nat. Prod.Rep., 19:49-59 (2002); and Raines, R. T. Protein Sci., 15:1219-1225(2006).

Further, it was previously shown that replacing Pro or Hyp in the Yaaposition with (2S,4R)-4-fluoroproline (Flp), first synthesized byGottleib et al., Biochemistry, 4:11:2507-2513 (1965), greatly increasestriple helix stability, see: U.S. Pat. No. 5,973,112; Holmgren et al.,Nature, 392:666-667 (1998); and Holmgren et al., Chem. Biol., 6:63-70(1999). In contrast, it has been shown that replacing Pro or Hyp in theYaa position with the diastereomer (2S,4S)-4-fluoroproline (flp) greatlydecreases stability, see: Bretscher et al., J. Am. Chem. Soc.,123:777-778 (2001). Accordingly, it is believed that a betterunderstanding of how the structure of collagen contributes to itsstability would facilitate the design of a collagen or collagen mimicswith improved stability.

A highly stable collagen substitute could advance the development ofimproved wound healing treatments. In recent years, there have beenexciting developments in wound healing, including the development oftissue engineering and tissue welding. For example, autologous epidermaltransplantation for the treatment of burns was a significant advance intissue engineering. Tissue engineering has also led to the developmentof several types of artificial skin, some of which employ human collagenas a substrate. However, a major problem associated with this treatmentis the fragile nature of these grafts during and after surgery.

Tissue welding is a wound healing technique in which a laser is used tothermally denature the collagen in the skin at the periphery of a wound.The wound is reannealed by permitting the renaturation of the collagen.In the case of large wounds, a “filler” or solder is required to effectreannealing of the wound. Various materials, including human albumin,have been used as solders for this purpose. A good solder is resilientand is non-immunogenic and should preferably be capable of interactionwith native collagen in adjacent sites.

Collagen is also used for a variety of other medical purposes. Forexample, collagen is used in sutures which can be naturally degraded bythe human body and thus do not have to be removed following recovery. Asometimes limiting factor in the design of collagen sutures is thestrength of the collagen fibers. A collagen variant or mimic having agreater strength would aid in the usage of such collagen sutures byrelieving this limitation. Accordingly, what is needed in the art is anovel collagen having increased stability for use in artificial skin, asa solder in tissue welding, and as a general tool for use in the designof medical constituents.

SUMMARY OF THE INVENTION

The present disclosure is summarized as novel variants of collagen,which have been designed to form a triple helix that is stronger thanthe native collagen. Specifically, collagen mimics are disclosed with atripeptide unit having the formula (Xaa-Yaa-Gly)_(n), where one of thepositions Xaa or Yaa is a bulky, non-electron withdrawing prolinederivative. By substituting a proline derivative at either the Xaa orYaa position in the native collagen helix, the stability of the helix isincreased due solely to steric effects relative to prior knowncollagen-related triple helices. Methods for making the novel collagenmimics are also described herein.

As such, a collagen mimic is disclosed having a tripeptide with theformula (Xaa-Yaa-Gly)_(n). This is best illustrated using generalstructural configuration found in FIG. 1. In the Xaa position, R2 is Hand R1 may be any bulky and non-electron withdrawing (or electrondonating substituent). Suitable R1 substituents may include but are notlimited to alkyl groups (methyl, ethyl, propyl, isopropyl or longeralkyls) and thiols groups; but, an electronegative atom such as N, O, F,Cl, or Br may not be installed directly on C4 of the proline ring.Likewise, in the Yaa position, R1 is H and R2 may include any bulky andnon-electron withdrawing (or electron donating substituent). Suitable R2substituents may include but are not limited to alkyl groups (methyl,ethyl, propyl, isopropyl or longer alkyls) and thiols groups; but, anelectronegative atom such as N, O, F, Cl, or Br may not be installeddirectly on C4 of the proline ring. The n is a positive integer.

Notably, in terms of stereochemistry, Xaa is a trans 4-substitutedproline and Yaa is a cis 4-substituted proline, wherein the substitutedgroup is a group that enforces pyrrolidine ring pucker via stericeffects resulting in a collagen triple helix that has increasedstability relative to the native collagen helix.

In one aspect, novel variants which endow structural stability tocollagen include a methyl proline group in one or both of the Xaa andYaa position of the triple helical collagen tripeptide having theformula (Xaa Yaa Gly)_(n). For example, in the Xaa position,(2S,4R)-4-methylproline, or mep, is used, and in the Yaa position, then(2S,4S)-4-methylproline, or Mep, is used. A tripeptide is present in thecollagen as at least one out of every three triplex repeats in thecollagen.

In a related aspect, a triple helix of collagen mimic molecules isdisclosed in which each of the molecules in the helix has a tripeptideformula of (flp-Yaa-Gly)_(n), where Yaa is (2S,4S)-4-methylproline, flpis (2S,4S)-4-fluoroproline, and n is a positive integer.

In a related aspect, a triple helix of collagen mimic molecules isdisclosed in which each of the molecules in the helix has a tripeptideformula of (Xaa-Flp-Gly)_(n), where Xaa is (2S,4R)-4-methylproline, andwhere Flp is (2S,4R)-4-fluoroproline, and n is a positive integer. Othersuitable substitutes for Flp can also include, for example, acetylmodified hydroxyproline, mesyl modified hydroxyproline, andtrifluoromethyl modified hydroxyproline.

In a related aspect, novel variants are disclosed which endow structuralstability to collagen by having a thioproline group in one or both ofthe Xaa and Yaa position of the triple helical collagen tripeptidehaving the formula (Xaa Yaa Gly)_(n). In the Xaa position,(2S,4R)-4-thioproline is used, and in the Yaa position, then(2S,4S)-4-thioproline is used.

In a related aspect, a triple helix of collagen mimic molecules isdisclosed in which each of the molecules in the helix has a tripeptideformula of (flp-Yaa-Gly)n, where Yaa is (2S,4S)-4-thioproline, flp is(2S,4S)-4-fluoroproline, and n is a positive integer.

In a related aspect, a triple helix of collagen mimic molecules isdisclosed in which each of the molecules in the helix has a tripeptideformula of (Xaa-Flp-Gly)n, where Xaa is (2S,4R)-4-thioproline, and whereFlp is (2S,4R)-4-fluoroproline, and n is a positive integer. Othersuitable substitutes for Flp can also include, for example, acetylmodified hydroxyproline, mesyl modified hydroxyproline, andtrifluoromethyl modified hydroxyproline.

It is an object of the present invention to provide a novel, highstability collagen molecule that could be used as a component inartificial skin, as a solder in tissue welding, or as a substitute forcollagen in other applications requiring high strength.

It is a feature of the present invention that evidence is provided todemonstrate the nature of the additional stability added to collagen bythe proline residue, thereby making it possible to design other residuesfor that position which would increase that stability.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused.

Other objects, advantages, and features of the present invention willbecome apparent upon review of the specification, drawings, and claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1. illustrates the pucker of the proline variants described hereand the ring conformations of 4-substituted proline residues. Suitablegroups for the R1 and R2 positions are described in the detaileddescription below.

FIG. 2 presents graphical data from the examples below. These data showconformational analysis of (mep-Pro-Gly)₇, (Pro-Mep-Gly)₇, and(mep-Mep-Gly)₇ by CD spectroscopy. FIG. 2A shows the spectra of peptidesolutions (0.2 mM in 50 mM acetic acid) incubated at less than or equalto 4° C. for more than 24 h. FIG. 2B shows the effect of temperature onthe molar ellipticity at 225 nm for (Pro-Mep-Gly)₇ and (mep-Mep-Gly)₇ or227 nm for (mep-Pro-Gly)₇. Data were recorded at intervals of 1 or 3° C.after equilibration for at least 5 min.

FIG. 3 shows a scheme for the synthesis of Fmoc-mep-Pro-GlyOH (6).

FIG. 4 shows a scheme for the synthesis of Fmoc-Pro-Mep-GlyOH (13).

FIG. 5 shows a scheme for the synthesis of Fmoc-mep-Mep-GlyOH (15).

FIG. 6 shows a scheme for the synthesis of Fmoc-(2S,4R)-thioproline(19), a thioproline derivative that can be used to incorporatethioprolines in collagen mimics via solid phase peptide synthesis in amanner analogous to that used to synthesize other collagen mimicsdescribed herein.

FIG. 7 is a table showing self-consistent field energies of Ac-Yaa-OMe.SCF energies (atomic units; au) of Ac-Yaa-OMe is calculated with B3LYPat 6-311+G(2d,p).

FIGS. 8A-C show circular dichroism spectral data for (mep-Pro-Gly)₇, (A)Circular dichroism spectra of (mep-Pro-Gly)₇ in the presence of TMAO(1.5, 2.0, 2.5, or 3.0 M) at 4° C. The maxima at ˜225 nm are indicativeof a collagen triple helix. (B) Thermal denaturation experiments with(mep-Pro-Gly)₇ in the presence of TMAO (1.5, 2.0, 2.5, or 3.0 M).Cooperative transitions are apparent in all four solutions. (C) Plot ofT_(m) values for (mep-Pro-Gly)₇ versus TMAO concentration. Linearregression and extrapolation to 0 M TMAO gives T_(m)=17.7° C.

FIG. 9 shows sedimentation equilibrium data. Sedimentation equilibriumdata for (Pro-Mep-Gly)₇ (red squares), (mep-Pro-Gly)₇ (blue squares) and(mep-Mep-Gly)₇ (black squares) at a rotor speed of 50,000 rpm.Equilibrium data were collected at 4° C. (filled squares) and 37° C.(open squares). Gradients were monitored at 230 nm. Best fits shown arefor solutions containing both trimer and some monomer at 4° C., and forsolutions containing only monomer at 37° C.

DETAILED DESCRIPTION OF THE INVENTION

The investigation that led to the work described here began with thenotion that a better understanding of the factors that contribute to thethree dimensional structure and stability of collagen would facilitatethe design of a collagen variant having improved strength for use inwound healing, and the development of treatments for people sufferingfrom collagen-related illnesses. It would also provide a stronger,general purpose collagen for a variety of uses.

Steric and stereoelectronic effects play a defining role in molecularconformation and reactivity. In small molecules, steric andstereoelectronic effects often have dichotomous consequences. Forexample, the anomeric effect in glycosides yields axial substituentsthat are disfavored by steric interactions. Similarly, replacing thesteric effect of a methyl group with the stereoelectronic effect of afluoro group enables a β-peptide to fold. It has been shown before thatstereoelectronic effects can be used to increase stability of collagenvariants over the natural collagen form. Here we show that stericeffects can be used to the same end.

In the native collagen polymer, the polyproline type II helices consistof over 300 repeats of the unit Xaa-Yaa-Gly, where Xaa is oftenS-proline (Pro) and the Yaa is often (2s,4r)-4-hydroxyproline (Hyp). Thepyrrolidine ring in the Xaa and Yaa positions have complementary puckerspreordained by a stereoelectronic effect. This stereoelectronic effectenhances the stability of collagen. One can achieve similarstereoelectronic effects substituting (2S,4S)-4-fluoroproline or “flp”for Xaa and (2S,4R)-4-fluoroproline or “Flp” for Yaa. Here we achievethat same level of stability using steric effects.

A broadly defined embodiment of the invention includes collagen mimicsthat contain one or more substitutions relative to the native collagenhelix formed of repeats of the tripeptide motif Pro-Hyp-Gly. The metesand bounds of this embodiment can be readily illustrated using FIG. 1,which shows the pucker of the proline variants described here and thering conformations of 4-substituted proline residues.

Accordingly, a collagen mimic is disclosed having a tripeptide with theformula (Xaa-Yaa-Gly)_(n). In the Xaa position, R2 is H and R1 may beany bulky and non-electron withdrawing (or electron donatingsubstituent). Suitable R1 substituents may include but are not limitedto alkyl groups (methyl, ethyl, propyl, isopropyl or longer alkyls) andthiols groups; but, an electronegative atom (N, O, F, Cl, Br) may not beinstalled directly on C4 of the proline ring. Likewise, in the Yaaposition, R1 is H and R2 may include any bulky and non-electronwithdrawing (or electron donating substituent). Suitable R2 substituentsmay include but are not limited to alkyl groups (methyl, ethyl, propyl,isopropyl or longer alkyls) and thiols groups; but, an electronegativeatom (N, O, F, Cl, Br) may not be installed directly on C4 of theproline ring. The n is a positive integer, suitably at least 3.

Notably, in one aspect of this embodiment, applicants identified thatthe pyrrolidine ring of (2S,4R)-4-methylproline (mep) has a strongpreference for a pucker matching that of (2S,4S)-4-fluoroproline, while(2S,4S)-4-methylproline (or Mep) has a strong preference for a puckermatching that of (2S,4R)-4-fluoroproline. Specifically, according toFIG. 1, the Cy-endo conformation is favored strongly by steric effectswhen R1=Me, R2=H (mep) or stereoelectronic effects when R1=H and R2=F(flp). Similarly, the Cy-exo conformation is favored strongly by stericeffects when R1=H, R2=CH3 (Mep) or stereoelectronic effects when R1=OH,R2=H (Hyp) or R1=F, R2=H (Flp). Thus, substituting one or both of mepand Mep into the appropriate positions in the collagen helix results ina collagen mimic of increased stability due solely to steric effects.

Accordingly, in one embodiment, collagen tripeptide mimics(Pro-Mep-Gly)₇, (mep-Pro-Gly)₇ and (mep-Mep-Gly)₇ are disclosed, whichare more stable than the native form of collagen. In a relatedembodiment, the results obtained from these methylproline tripeptideswere combined with applicants prior work (i.e., disclosing the use of(2S,4S)-4-fluoroproline (flp) in the first position and(2S,4R)-4-fluoroproline (Flp) in the second position) to discover thatflp-Mep-Gly and mep-Flp-Gly offer similar advantages over naturalcollagen. In fact, the variants flp-Mep-Gly and mep-Flp-Gly exhibitedhigher melting temperatures that any of the other known collagen mimics.

Applicants note that not every variant in this motif yields structureswith added stability. For example, a collagen mimic constructed with themotif flp-Flp-Gly turned out to yield relatively unstable collagenvariants, even though the variants with the fluoroproline in only oneposition had improved stability. The explanation for this result seemsto be steric hindrance between the fluorine atoms. The methyl groups inthe methylproline variants taught here do not have the same problem asthese groups are positioned extending (jut out) radially from the axisof the collagen tripeptide helix and hence they do not interfere witheach other. As such, it is reasonable to expect that the methyl groupsin the methylproline variants could be substituted by other alkyl orfunctional groups to achieve the same effect, in the same manner as themethyl groups.

In one embodiment, a thiol group can be substituted for a methyl groupin the collagen tripeptide as sulfur is also a bulky electron donatingsubstituent. A thiol group behaves like a methyl, as sulfur has a largesize and a similar electronegativity to carbon. e sulfur of a thiolgroup, like the carbon of a methyl group, has only modestelectronegativity (2.5 on the Pauling scale) and hence is expected toexert its effects by steric rather than stereoelectronic effects.

Accordingly, as used herein the abbreviation “thp” refers to(2S,4R)-thioproline. Similarly, (2S,4S)-thioproline can be abbreviatedas “Thp”. These thioprolines may be incorporated as an amino acidderivative into a collagen tripeptide to improve its stability relativeto the native collagen tripeptide. Suitable tripeptides include, forexample, (thp-Thp-Gly)₇, (thp-Mep-Gly)₇, (mep-Thp-Gly)₇, (Pro-Thp-Gly)₇,(thp-Pro-Gly)₇, (thp-Hyp-Gly)₇, (flp-Thp-Gly)₇, and (thp-Flp-Gly) 7.

It is also contemplated that the use of the steric and stereoelectroniceffects can be combined. For example, one could install a proline at thefirst (Yaa) position with both a fluoro and a methyl group attached tothe C4, with the fluoro group in the 4(R) configuration and the methylgroup in the 4(S) configuration. Similar variants, with the appropriateconfiguration, can be envisioned at the Xaa position, again providedonly that the variants which reduce stability (fluoro groups interferingbetween Xaa and Yaa) are avoided.

Thus, it is envisioned here that the mep and Mep containing collagenconstituents can be used in collagen mimics with other prolinederivatives at the other position. As used herein, the term “prolinederivatives” is intended to include but is not limited to Hyp or(2S,4R)-4-hydroxyproline, flp or (2S,4S)-4-fluoroproline, Flp or(2S,4R)-4-fluoroproline, mep or (2S,4S)-4-methylproline or, Mep or(2S,4R)-4-methylproline, Thp or (2S,4R)-4-thioproline, thp or(2S,4S)-4-thioproline, and other longer alkyl proline derivativesdescribed herein. Such proline derivatives are bulky in size and have asimilar electronegativity as carbon, but are not prevented by sterichindrance from incorporation into a collagen triple helical strand.

As used herein, the term “alkyl proline derivatives” is intended toinclude but is not limited to derivatives of proline where thefunctional group is 4-methyl, 4-ethyl, 4-propyl, 4-isopropyl, and otherperhaps longer alkyl groups with similar electron donating substituents.Suitable alkyl proline derivatives enforce ring pucker via the stericeffect and should thus be stabilizing to collagen triple helices.Notably, the thiol groups are also encompassed within this functionaldefinition, given that sulfur and carbon have similarelectronegativities and that sulfur is much larger (bulkier) thancarbon.

It is also envisioned that stronger collagen polymers can be made usingthe techniques described here even if the triplex motifs described hereonly make up a portion of the triplexes in the collagen monomers. Forexample, a collagen monomer could beAaa-Baa-Gly-Xaa-Yaa-Gly-Aaa-Bbb-Gly, where Aaa is Pro, Baa is Mep, Xaais Pro and Yaa is Hyp (or Flp). Or the Mep and Hyp could be reversed. Itis envisioned that many such arrangements of triplex combinations arepossible.

It is to be understood that this invention is not limited to theparticular methodology, protocol, and reagents described, and as suchmay vary. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

EXAMPLES Example 1 Experimental Materials and Methods

Commercial chemicals were of reagent grade or better, and were usedwithout further purification. Anhydrous THF, DMF, and CH₂Cl₂ wereobtained from CYCLE-TAINER® solvent delivery systems (J. T. Baker,Phillipsburg, N.J.). Other anhydrous solvents were obtained inseptum-sealed bottles. In all reactions involving anhydrous solvents,glassware was either oven- or flame-dried. NaHCO₃ and brine (NaCl) referto saturated aqueous solutions unless specified otherwise. Flashchromatography was performed with columns of silica gel 60, 230-400 mesh(Silicycle, Quebec City, Canada). Semi-preparative HPLC was performedwith a Zorbax C-8 reversed-phase column. Analytical HPLC was performedwith an Agilent C-8 reversed-phase column using linear gradients ofsolvent A (H₂O with 0.1% v/v TFA) and solvent B (CH₃CN with 0.1% v/vTFA).

The term “concentrated under reduced pressure” refers to the removal ofsolvents and other volatile materials using a rotary evaporator at wateraspirator pressure (<20 torr) while maintaining the water-bathtemperature below 40° C. Residual solvent was removed from samples athigh vacuum (<0.1 torr). The term “high vacuum” refers to vacuumachieved by a mechanical belt-drive oil pump.

NMR spectra were acquired with a Bruker DMX-400 Avance spectrometer (¹H,400 MHz; ¹³C, 100.6 MHz) at the National Magnetic Resonance Facility atMadison (NMRFAM). NMR spectra were obtained at ambient temperatures onsamples dissolved in CDCl₃ unless indicated otherwise. Couplingconstants J are provided in Hertz. Compounds with a carbamate protectinggroup (e.g., Boc or Fmoc) exist as mixtures of Z and E isomers that donot interconvert on the NMR time scale at ambient temperatures.Accordingly, these compounds exhibit two sets of NMR signals, exceptwhen spectra are obtained at higher temperature (as indicated).

Mass spectrometry was performed with either a Micromass LCT(electrospray ionization, ESI) in the Mass Spectrometry Facility in theDepartment of Chemistry or an Applied Biosystems Voyager DE-Pro(matrix-assisted laser desorption/ionization, MALDI) mass spectrometerin the University of Wisconsin Biophysics Instrumentation Facility.

Example 2 Synthesis of N-tert-Butyloxycarbonyl-(2S,4R)-4-methylproline(1) and(S)-2-tert-butyldimethylsilyloxymethyl-N-tert-butyloxycarbonyl-4-methylenepyrrolidine(7)

These compounds were synthesized by the method of Del Valle and GoodmanM. J. Org. Chem. 2003, 68, 3923-3931.N-tert-Butyloxycarbonyl-S-prolyl-glycine benzyl ester (4) wassynthesized by the method of Jenkins et al., T. Org. Lett. 2005, 7,2619-2622. Synthetic routes toN-(9-fluorenylmethoxycarbonyl)-(2S,4R)-4-methylprolyl-S-prolyl-glycine 6and N-(2-¹³CH₃-acetyl)-(2S,4R)-4-methylproline methyl ester (2),N-(9-fluorenylmethoxycarbonyl)-(2S)-prolyl-(2S,4S)-4-methylprolyl-glycine(13) and N-(2-¹³CH₃-acetyl)-(2S,4S)-4-methylproline methyl ester (10),andN-(9-fluorenylmethoxycarbonyl)-(2S,4R)-4-methylprolyl-(2S,4S)-4-methylprolyl-glycine(15) are summarized in FIGS. 3-5, respectively. The synthesis forcompounds 16-19 are summarized in FIG. 6.

Example 3 Synthesis of N-(2-¹³CH₃-Acetyl)-(2S,4R)-4-methylproline methylester (2)

Following the method of Nudelman et al, Synth. Commun. 1998, 28,471-474, compound 1 (80 mg, 0.35 mmol) was dissolved in anhydrous MeOH(11 mL), and the resulting solution was cooled to 0° C. Acetyl chloride(12.1 g, 150 mmol) was added dropwise and the reaction mixture wasallowed to warm slowly to room temperature and stirred for 6 h. Theresulting solution was concentrated under reduced pressure and theresidue dissolved in anhydrous CH₂Cl₂ (15 mL).N,N-4-Dimethylaminopyridine (385 mg, 3.2 mmol) was added, followed bythe dropwise addition of H₃ ¹³CC(O)Cl (250 mg, 3.0 mmol) and thereaction mixture was stirred for 24 h. MeOH (5 mL) was added to quenchthe reaction. The resulting solution was concentrated under reducedpressure, and the residue was dissolved in 10% w/v aqueous citric acid,extracted with CH₂Cl₂ (2×40 mL), dried over anhydrous MgSO₄(s), andconcentrated under reduced pressure. The crude product was purified byflash chromatography (50% v/v EtOAc in hexane to elute byproductsfollowed by 6% v/v MeOH in EtOAc) to afford 2 (50 mg, 0.27 mmol, 77%) asa colorless oil. ¹H NMR δ: 1.08 and 1.07 (2 d, J=5.8, 3H), 1.76-1.86 (m,1H), 2.08 (d, J_(C-H)=128, 3H), 2.06-2.09 (m, 1H), 2.52 (m, 1H), 3.05(t, J=9.2, 1H), 3.71-3.83 (m, 4H), 4.41 and 4.54 (2 dd, J=2.4, 9.0, 1H);¹³C NMR δ: 17.5, 17.9, 22.1, 22.3, 32.7, 37.2, 52.4, 53.3, 54.9, 58.7,60.7, 169.3, 169.8, 173.0; HRMS-ESI (m/z): [M+Na]⁺ calcd for C₈¹³CH₁₅NO₃Na, 209.0983; found, 209.0987.

Example 4 Synthesis ofN-(9-Fluorenylmethoxycarbonyl)-(2S,4R)-4-methylproline (3)

Compound 1 (0.88 g, 3.8 mmol) was dissolved in 4 N HCl in dioxane (20mL) under Ar(g) and stirred for 1.5 h. The resulting solution wasconcentrated under reduced pressure and the residue dissolved in dioxaneand concentrated under reduced pressure again. The resulting free aminewas dissolved in 10% w/v aqueous NaHCO₃ (55 mL), and a solution ofFmoc-OSu (1.41 g, 4.2 mmol) in dioxane (18 mL) was added. Additionaldioxane (40 mL) was added, and the resulting white suspension stirredfor 27 h. The dioxane was removed under reduced pressure and the aqueoussolution was diluted with water (100 mL) and washed with ether (4×60mL). The aqueous layer was acidified to pH 1.5 with 2 M HCl, extractedwith EtOAc (4×160 mL), dried over anhydrous MgSO₄(s), and concentratedunder reduced pressure to afford 3 (1.11 g, 3.2 mmol, 87%) as a whitesolid. ¹H NMR δ: 1.04 and 1.08 (d, J=6.5, 3H), 1.73-1.94 (m, 1H),2.11-2.20 and 2.31-2.50 (m, 2H), 2.97-3.05 (m, 1H), 3.68-3.79 (m, 1H),4.11-4.52 (m, 4H), 7.24-7.45 (m, 4H), 7.51-7.63 (m, 2H), 7.68-7.81 (m,2H); ¹³C NMR δ: 17.3, 31.2, 32.4, 36.7, 38.7, 47.1, 47.3, 53.6, 53.8,58.9, 59.8, 66.0, 67.5, 68.2, 120.1, 120.2, 125.0, 125.2, 125.2, 127.2,127.2, 127.8, 127.9, 141.5, 143.8, 143.9, 156.4, 175.3, 177.8; ESI-MS(m/z): [M−H]⁻ calcd for C₂₁H₂₀NO₄Na, 350.1; found, 350.6.

Example 5 Synthesis ofN-9-Fluorenylmethoxycarbonyl)-(2S,4R)-4-methylprolyl-S-prolyl-glycinebenzyl ester (5)

Compound 4 (1.34 g, 3.8 mmol) was dissolved in 4 N HCl in dioxane (27mL) under Ar(g) and stirred for 1.4 h. The resulting solution wasconcentrated under reduced pressure and the residue was dissolved inanhydrous CH₂Cl₂ (30 mL) and cooled to 0° C. Compound 3 (430 mg, 1.3mmol) was added to the solution, followed by PyBroP (606 mg, 1.3 mmol)and DIEA (1.26 g, 9.8 mmol). The reaction mixture was allowed to warmslowly to room temperature, stirred for 36 h, and then washed with 10%w/v aqueous citric acid (100 mL), NaHCO₃ (100 mL), water (100 mL), andbrine (100 mL), dried over anhydrous MgSO₄(s), and concentrated underreduced pressure. The crude product was purified by flash chromatography(gradient: 100% hexane to 100% EtOAc) to afford 5 (442 mg, 0.7 mmol,57%) as a white solid containing an unidentified impurity which wasremoved after the succeeding step. HRMS-ESI (m/z): [M+Na]⁺ calcd forC₃₅H₃₇N₃O₆Na, 618.2580; found, 618.2594.

Example 6 Synthesis ofN-9-Fluorenylmethoxycarbonyl-(2S,4R)-4-methylprolyl-S-prolyl-glycine (6)

MeOH (50 mL) was added carefully to a mixture of compound 5 (420 mg, 0.7mmol) and Pd/C (10% w/w, 90 mg, 0.1 mmol) under Ar(g), and the resultingblack suspension was stirred under H₂(g) for 5 h. Careful monitoring byTLC was necessary to prevent hydrogenolysis of the Fmoc group. Thesuspension was filtered through a pad of Celite and concentrated underreduced pressure. The crude product was purified by flash chromatography(EtOAc to elute byproducts, then 25% v/v MeOH in CH₂Cl₂ with 0.1% formicacid). The fractions containing 6 were concentrated under reducedpressure, and the formic acid was removed by dissolving the residue in10% v/v MeOH in toluene and concentrating under reduced pressure toafford 6 (300 mg, 0.6 mmol, 84%) as a white solid. The purity of 6 wasdetermined to be 90% by analytical HPLC (gradient: 15% B to 85% B over50 min). A small sample was purified further by semi-preparative HPLCfor use in NMR experiments. ¹H NMR (spectrum obtained at 343 K inDMSO-d₆) δ: 1.06-0.93 (m, 3H), 1.7-2.47 (m, 7H), 2.81-3.75 (m, 6H),4.11-4.58 (m, 5H), 7.26-7.46 (m, 4H), 7.50-7.92 (m, 5H); ¹³C NMR(DMSO-d₆) δ: 17.4, 17.5, 24.3, 24.3, 28.9, 29.0, 30.0, 31.3, 36.3, 37.2,46.1, 46.5, 46.6, 46.9, 53.1, 53.7, 57.6, 58.0, 59.1, 59.2, 66.0, 66.5,120.0, 120.1, 124.8, 124.9, 125.1, 127.1, 127.2, 127.6, 127.7, 140.7,143.9, 144.0, 153.6, 153.8, 158.1, 158.4, 158.8, 169.9, 170.0, 171.2,171.8, 172.0; HRMS-ESI (m/z): [M+Na]⁺ calcd for C₂₈H₃₁N₃O₆Na, 528.2111;found, 528.2108.

Example 7 Synthesis ofN-tert-Butyloxycarbonyl-(2S,4S)-2-hydroxymethyl-4-methylpyrrolidine (8)

Following the method of Del Valle and Goodman, a 0.1 M solution ofcompound 7 (9.78 g, 29.8 mmol) in MeOH was prepared. Raney-nickel (˜1.00g) that had been washed repeatedly with MeOH was added to the solution,and the flask was flushed repeatedly with H₂(g). The solution wasstirred under H₂(g) for 30 h and then filtered through a Celite pad.(Caution: do not Allow the Filter Cake to Dry During Filtration asRaney-Nickel can Rapidly Ignite.) The resulting solution wasconcentrated under reduced pressure and then dissolved in 0.5 M TBAF inTHF (120 mL). After stirring for 15 h, the solution was concentratedunder reduced pressure. The synthetic procedure was expected to yieldboth 8 and its trans diastereomer in a 3:1 ratio. The major diastereomer8 was purified by flash chromatography (7% v/v EtOAc in hexane).Compound 8 was obtained (3.82 g, 17.7 mmol, 59% (2 steps)) as acolorless oil. The ratio of 8 to its 4R diastereomer was determined tobe 30:1 by gas chromatography with a Supelco β-Dex-250 chiral column (17m) and N₂ as the carrier gas at a column temperature of 110° C. ¹H NMRδ: 1.02 (d, J 6.1, 3H), 1.08 (m, 1H), 1.47 (s, 9H), 2.04-2.21 (m, 2H),2.77 (t, J=10.1, 1H), 3.53-3.73 (m, 3H), 3.82-3.98 (m, 1H), 5.33 (d,J=8.4, 1H); ¹³C NMR δ: 17.0, 28.5, 28.7, 37.5, 54.5, 55.0, 61.6, 67.9,80.4, 157.1; ESI-MS (m/z): [M+Na]⁺ calcd for C₁₁H₂₁NO₃Na, 238.1; found,238.3.

Example 8 Synthesis of N-tert-Butyloxycarbonyl-(2S,4S)-4-methylproline(9)

Following the method of Del Valle and Goodman, three solutions wereprepared prior to the oxidation. The first solution consisted of NaClO₂(1.33 g, 14.7 mmol) in water (7.4 mL). The second solution consisted ofbleach (436 μL) in water (7.4 mL). The third solution consisted ofcompound 8 (1.60 g, 7.4 mmol) dissolved in 100 mL of 3:2 CH₃CN:NaH₂PO₄buffer (pH 6.6, 0.67 M). The solution containing 8 was heated to 45° C.,and TEMPO (193 mg, 0.7 mmol) was added. The two oxidant solutions wereadded simultaneously in 618 μL portions over 1 h, and the resultingsolution was stirred at 40° C. for 18 h. After cooling to roomtemperature, the reaction was quenched by dropwise addition of saturatedaqueous Na₂SO₃ until the solution became colorless. The acetonitrile wasremoved under reduced pressure, and the resulting aqueous solutionbasified to pH 10 with 1 M NaOH. The basic solution was washed withether (5×125 mL) and then acidified to pH 2 with 2 M HCl. The acidicsolution was extracted with ether (4×200 mL), and the organic layer wasdried over anhydrous MgSO₄(s) and concentrated under reduced pressure toafford 9 (1.60 g, 7.0 mmol, 94%) as a white solid. ¹H NMR δ: 1.09 (d,J=6.0, 3H), 1.44 and 1.50 (s, 9H), 1.58-1.70 and 1.88-2.00 (m, 1H),2.21-2.31 (m, 1H), 2.31-2.52 (m, 1H), 2.89-3.04 (m, 1H), 3.67-3.82 (m,1H), 4.20-4.38 (2m, 1H); ¹³C NMR δ: 16.9, 17.2, 28.2, 28.3, 32.7, 36.4,38.8, 53.3, 54.1, 59.4, 59.5, 80.4, 81.6, 159.4, 162.1, 174.9, 179.6;ESI-MS (m/z): [M−H]⁻ calcd for C₁₁H₁₈NO₄, 228.1; found, 228.4.

Example 9 Synthesis of N-(2-¹³CH₃-Acetyl)-(2S,4S)-4-methylproline methylester (10)

Following the method of Nudelman et al., compound 9 (100 mg, 0.44 mmol)was dissolved in anhydrous MeOH (10 mL), and the resulting solution wascooled to 0° C. Acetyl chloride (11.80 g, 150 mmol) was added dropwiseand the reaction mixture was allowed to warm slowly to room temperatureand stirred for 10 h. The resulting solution was concentrated underreduced pressure and the residue dissolved in anhydrous CH₂Cl₂ (15 mL).N,N-4-Dimethylaminopyridine (450 mg, 3.7 mmol) was added, followed bythe dropwise addition of H₃ ¹³CC(O)Cl (99 mg, 1.2 mmol). The reactionmixture was stirred for 9 h. Additional unlabeled acetyl chloride wasadded to ensure complete reaction, followed by MeOH (10 mL) to quenchthe reaction. The resulting solution was concentrated under reducedpressure, and the residue was dissolved in 10% w/v aqueous citric acid,extracted with CH₂Cl₂ (2×40 mL), dried over anhydrous MgSO₄(s), andconcentrated under reduced pressure. The crude product was purified byflash chromatography (50% v/v EtOAc in hexane to elute byproductsfollowed by 6% v/v MeOH in EtOAc) to afford 10 (40 mg, 0.21 mmol, 52%)as a yellow oil. ¹H NMR δ: 1.06 and 1.10 (2 d, J=6.4, 3H), 1.56 (q,J=10.5, 1H), 2.09 (d, J_(C-H)=128, 3H), 2.28-2.46 (m, 2H), 3.18 (t,J=9.8, 1H), 3.69 (m, 1H), 3.74 and 3.78 (2 s, 3H), 4.36 (t, J=8.4, 1H);¹³C NMR δ: 17.0, 21.8, 22.4, 33.9, 37.6, 52.3, 55.1, 59.3, 168.9, 169.4,173.1, 173.2; HRMS-ESI (m/z): [M+Na]⁺ calcd for C₈ ¹³CH₁₅NO₃Na,209.0983; found, 209.0980.

Example 10 Synthesis ofN-tert-Butyloxycarbonyl-(2S,4S)-4-methylprolyl-glycine benzyl ester (11)

Compound 9 (1.6 g, 7.0 mmol), glycine benzyl ester tosylate (3.07 g, 9.1mmol), and PyBOP (3.64 g, 7.0 mmol) were dissolved in anhydrous CH₂Cl₂(80 mL). DIEA (2.26 g, 17.5 mmol) was added, and the resulting solutionwas stirred for 27 h under Ar(g). The reaction mixture was washed with10% w/v aqueous citric acid (3×50 mL), NaHCO₃ (3×50 mL), water (50 mL),and brine (50 mL), dried over anhydrous MgSO₄(s), and concentrated underreduced pressure. The crude oil was purified by flash chromatography(1:1 EtOAc:hexane) to afford 11 (2.13 g, 5.9 mmol, 84%) as a colorless,sticky liquid. ¹H NMR δ: 1.03 and 1.04 (d, J=3.2, 3H), 1.44 (bs, 9H),1.55-2.50 (m, 4H), 2.90 (t, J=9.8, 1H), 3.65-3.94 (m, 1H), 4.01-4.34 (m,3H), 5.18 (s, 2H), 7.36 (bs, 5H); HRMS-ESI (m/z): [M+Na]⁺ calcd forC₂₀H₂₈N₂O₅Na, 399.1896; found, 399.1897.

Example 11 Synthesis ofN-9-Fluorenylmethoxycarbonyl-S-prolyl-(2S,4S)-4-methylprolyl-glycinebenzyl ester (12)

Compound 11 (1.18 g, 3.3 mmol) was dissolved in 4 N HCl in dioxane (30mL) under Ar(g) and stirred for 2.5 h. The resulting solution wasconcentrated under reduced pressure and the residue dissolved inanhydrous DMF (50 mL). DIEA (1.60 g, 12.2 mmol) was added, followed byFmoc-Pro-OPfp (3.52 g, 7.0 mmol), and additional anhydrous DMF (20 mL).The solution was stirred for 48 h and then concentrated by rotaryevaporation under high vacuum. Flash chromatography (gradient: 25% v/vEtOAc in hexane to 95% v/v EtOAc in hexane) afforded 12 (800 mg, 1.3mmol, 40%) as a white solid. ¹H NMR δ: 1.04 and 1.07 (d, J=6.5, 3H),1.76-2.60 (m, 8H), 3.44-3.75 (m, 2H), 3.91-4.61 (m, 8H), 5.27 (s, 2H),7.04-7.79 (m, 13H); HRMS-ESI (m/z): [M+Na]⁺ calcd for C₃₅H₃₇N₃O₆Na,618.2580; found, 618.2558.

Example 12N-9-Fluorenylmethoxycarbonyl-S-prolyl-(2S,4S)-4-methylprolyl-glycine(13)

MeOH (130 mL) was added carefully to a mixture of compound 12 (800 mg,1.3 mmol) and Pd/C (10% w/w, 160 mg, 0.2 mmol) under Ar(g), and theresulting black suspension was stirred under H₂(g) for 2 h. Carefulmonitoring by TLC was necessary to prevent hydrogenolysis of the Fmocgroup. The suspension was filtered through a pad of Celite andconcentrated under reduced pressure. The crude product was purified byflash chromatography (EtOAc to elute byproducts, then 12% v/v MeOH inCH₂Cl₂ containing 0.1% v/v formic acid). The fractions containing 13were concentrated under reduced pressure and the formic acid was removedby dissolving the residue in 10% v/v MeOH in toluene and concentratingunder reduced pressure to afford 13 (500 mg, 1.0 mmol, 73%) as a whitesolid. The purity of 13 was determined to be 90% by analytical HPLC(gradient: 15% B to 85% B over 50 min). ¹H NMR (spectrum obtained at 343K in DMSO-d₆) δ: 1.02 (d, J=6.5, 3H), 1.37-1.49 (m, 1H). 1.70-2.00 (m,3H), 2.06-2.27 (m, 2H), 2.87-3.46 (m, 6H), 3.74-4.02 (m, 3H), 4.11-4.57(m, 5H), 7.25-7.90 (m, 8H); ¹³C NMR (DMSO-d₆) δ: 16.8, 22.6, 23.7, 28.5,29.3, 33.3, 33.4, 37.0, 37.1, 40.7, 46.2, 46.6, 46.8, 46.9, 53.6, 53.8,57.6, 58.0, 59.8, 59.9, 66.3, 66.5, 120.1, 120.2, 125.0, 125.1, 125.1,125.3, 127.1, 127.1, 127.2, 127.3, 127.7, 140.7, 140.7, 143.8, 143.9,144.0, 153.8, 153.8, 169.5, 169.6, 171.2, 171.7, 171.7; HRMS-ESI (m/z):[M−H]⁻ calcd for C₂₈H₃₀N₃O₆, 504.2135; found, 504.2121.

Example 13 Synthesis ofN-9-Fluorenylmethoxycarbonyl-(2S,4R)-4-methylprolyl-(2S,4S)-4-methylprolyl-glycinebenzyl ester (14)

Compound 11 (980 mg, 2.7 mmol) was dissolved in 4 N HCl in dioxane (30mL) under Ar(g) and stirred for 1.7 h. The reaction mixture wasconcentrated under reduced pressure and the residue was dissolved inanhydrous CH₂Cl₂ (80 mL) and cooled to 0° C. Compound 3 (430 mg, 1.3mmol) was added to the solution, followed by PyBroP (653 mg, 1.4 mmol)and DIEA (1.00 g, 7.8 mmol). The resulting solution was allowed to warmslowly to room temperature and then stirred for 40 h. The reactionmixture was diluted with CH₂Cl₂ (125 mL), washed with 10% w/v aqueouscitric acid (100 mL), NaHCO₃ (100 mL), water (100 mL), and brine (100mL), dried over anhydrous MgSO₄(s), and concentrated under reducedpressure. Flash chromatography (gradient: 35% v/v EtOAc in hexane to 90%v/v EtOAc in hexane) afforded 14 (520 mg, 0.9 mmol, 66%) as a whitesolid. ¹H NMR δ: 0.96-1.30 (m, 6H), 1.63-3.13 (m, 9H), 3.69-4.66 (m,7H), 5.15 (m, 2H), 7.14-7.79 (m, 13H); HRMS-ESI (m/z): [M+Na]⁺ calcd forC₃₆H₃₉N₃O₆Na, 632.2737; found, 632.2712.

Example 14 Synthesis ofN-9-Fluorenylmethoxycarbonyl-(2S,4R)-4-methylprolyl-(2S,4S)-4-methylprolyl-glycine(15)

MeOH (60 mL) was added carefully to a mixture of compound 14 (520 mg,0.9 mmol) and Pd/C (10% w/w, 160 mg, 0.2 mmol) under Ar(g), and theresulting black suspension was stirred under a hydrogen atmosphere for2.5 h. Careful monitoring by TLC was necessary to prevent hydrogenolysisof the Fmoc group. The suspension was filtered through a pad of Celiteand concentrated under reduced pressure. The crude product was purifiedby flash chromatography (EtOAc to elute byproducts, then 12% v/v MeOH inCH₂Cl₂ with 0.1% formic acid). The fractions containing 15 wereconcentrated under reduced pressure, and the formic acid was removed bydissolving the residue in 10% v/v MeOH in toluene and concentratingunder reduced pressure to afford 15 (315 mg, 0.6 mmol, 66%) as a whitesolid. The purity of 15 was determined to be 90% by analytical HPLC(gradient 15% B to 85% B over 50 min). ¹H NMR (spectrum obtained at 343K in DMSO-d₆) δ: 0.9-1.05 (m, 6H), 1.27 (m, 1H), 1.42 (m, 1H), 1.72-1.82(m, 1H), 2.01-2.35 (m, 4H), 2.84-3.00 (m, 3H), 3.48-3.66 (m, 3H),3.70-3.80 (m, 1H), 4.23-4.59 (m, 4H), 7.29-7.45 (m, 4H), 7.52-7.77 (m,2H), 7.84-7.91 (m, 2H); ¹³C NMR (DMSO-d₆) δ: 14.0, 16.8, 17.3, 17.5,17.6, 18.2, 22.1, 30.0, 30.5, 31.0, 31.2, 31.2, 33.3, 33.4, 33.8. 35.3.36.1. 37.0. 46.6, 47.0, 51.3, 51.6, 53.0, 53.6, 53.8, 57.9, 58.1, 58.4,59.8, 59.9, 60.0, 66.2, 66.5, 109.6, 120.1, 120.2, 121.4, 124.9, 125.1,125.1, 127.1, 127.1, 127.2, 127.3, 127.7, 127.7, 128.9, 140.7, 140.7,143.8, 152.4, 153.8, 169.4, 169.5, 171.3, 171.5, 171.6; HRMS-ESI (m/z):[M−H]⁻ calcd for C₂₉H₃₃N₃O₆, 518.2291; found, 518.2307.

Example 15 Measurement of K_(trans/cis) Values of (2) and (10)

Each compound (5-10 mg) was dissolved in D₂O with enough CD₃OD added tosolubilize the compound (less than 20% of total volume). The ¹³C NMRspectra were recorded using an inverse gated decoupling pulse programwith a relaxation delay of 100 s and a pulse width of 10 μs. Thespectral baselines were corrected and peaks corresponding to the labeledcarbon were integrated with the software package NUTS-NMR UtilityTransform Software, Acorn NMR, Inc., 7670 Las Positas Road, Livermore,Calif. 94551. Values of K_(trans/cis) were determined by the relativeareas of the trans and cis peaks for the labeled carbons.

Example 16 Attachment of Fmoc-mep-Pro-GlyOH (6) to 2-Chlorotrityl Resin

Under Ar(g), 33 mg (0.053 mmol) of 2-chlorotrityl resin (loading: 1.6mmol/g) was swelled in anhydrous CH₂Cl₂ (0.7 mL) for 5 min. A solutionof compound 6 (25 mg, 0.050 mmol) and DIEA (26 mg, 0.20 mmol) inanhydrous CH₂Cl₂ (0.7 mL) was added by syringe. Additional anhydrousCH₂Cl₂ (0.5 mL) was used to ensure complete transfer of 6. After 2 h,anhydrous MeOH (0.2 mL) was added to cap any remaining active sites onthe resin. The resin-bound peptide was isolated by gravity filtration,washed with several portions of anhydrous CH₂Cl₂ (˜25 mL), and driedunder high vacuum. The mass of the resin-bound peptide was 57 mg.Loading was measured by ultraviolet spectroscopy to be 0.69 mmol/g (see,Applied Biosystems Determination of the Amino Acid Substitution Levelvia an Fmoc Assay; Technical Note 123485 Rev 2; Documents onDemand-Applied Biosystems Web Page,http://docs.appliedbiosystems.com/search.taf (Nov. 30, 2005)).

Example 17 Attachment of Fmoc-Pro-Mep-GlyOH (13) and Fmoc-mep Mep-GlyOH(15) to 2-Chlorotrityl Resin

Fmoc-tripeptides 13 and 15 were loaded onto 2-chlorotrityl resin insimilar fashion to that described for 6. Loadings were measured byultraviolet spectroscopy^(S5) to be 0.56 mmol/g for 13 and 0.60 mmol/gfor 15.

Example 18 Synthesis of (mep-Pro-Gly)₇, (Pro-Mep-Gly)₇, and(mep-Mep-Gly)₇

These three 21-mer peptides were synthesized by segment condensation oftheir corresponding Fmoc-tripeptides (6, 13, and 15) on solid phaseusing an Applied Biosystems Synergy 432A Peptide Synthesizer at theUniversity of Wisconsin-Madison Biotechnology Center. The first trimerwas loaded onto the resin as described above. Fmoc-deprotection wasachieved by treatment with 20% (v/v) piperidine in DMF. The trimers (3equivalents) were converted to active esters by treatment with HBTU,DIEA, and HOBt. Extended couplings (120-200 min) were employed at roomtemperature.

Peptides were cleaved from the resin in 95:3:2TFA:triisopropylsilane:H₂O (1.5 mL), precipitated fromt-butylmethylether at 0° C., and isolated by centrifugation.Semi-preparative HPLC was used to purify the peptides (mep-Pro-Gly)₇(gradient: 10% B to 40% B over 50 min), (Pro-Mep-Gly)₇ (gradient: 15% Bto 50% B over 50 min), and (mep-Mep-Gly)₇ (gradient: 15% B to 60% B over60 min). All three peptides were >90% pure by analytical HPLC andMALDI-TOF mass spectrometry (m/z) [M+H]⁺ calcd for C₉₁H₁₃₆N₂₁O₂₂ 1876.2.found 1875.6 for (mep-Pro-Gly)₇, 1875.4 for (Pro-Mep-Gly)₇. calcd forC₉₈H₁₅₀N₂₁O₂₂ 1974.4. found 1973.7 for (mep-Mep-Gly)₇.

Example 19 Circular Dichroism Spectroscopy of (mep-Pro-Gly)₇,(Pro-Mep-Gly)₇, and (mep-Mep-Gly)₇

Peptides were dried under vacuum for at least 24 h before being weighedand dissolved to 0.2 mM in 50 mM acetic acid (pH 2.9). The solutionswere incubated at <4° C. for >24 h before CD spectra were acquired usingan Aviv 202SF spectrometer at the University of Wisconsin BiophysicsInstrumentation Facility. Spectra were measured with a 1-nm band-pass incuvettes with a 0.1-cm pathlength. The signal was averaged for 3 sduring wavelength scans and either 5 or 15 s during denaturationexperiments. During denaturation experiments, CD spectra were acquiredat intervals of 1° C. for (mep-Pro-Gly)₇ and 3° C. for (Pro-Mep-Gly)₇and (mep-Mep-Gly)₇. At each temperature, solutions were equilibrated fora minimum of 5 min before data acquisition. Values of T_(m) weredetermined by fitting molar ellipticity at 225 nm (for (Pro-Mep-Gly)₇and (mep-Mep-Gly)₇) or 227 nm (for (mep-Pro-Gly)₇) to a two-state model.(See, Becktel, W. J.; Schellman, J. A. Biopolymers 1987, 26, 1859-1877).T_(m) values were determined in triplicate.

Example 20 Circular Dichroism Spectroscopy of (mep-Pro-Gly)₇ inSolutions Containing Trimethylamine-N-Oxide

(mep-Pro-Gly)₇ was dried under vacuum for 24 h before being weighed anddissolved to 0.2 mM in solutions of 50 mM acetic acid containing 1.5,2.0, 2.5, or 3.0 M trimethylamine-N-oxide (TMAO), respectively.(Solution pH was corrected to pH=2.9 by addition of concentrated HCl.)Solutions were incubated at ≦4° C. for ≧24 h before CD spectra wererecorded using the methods described in the previous section. FIGS. 8Aand 8B show the CD spectra and the thermal melts for each solution. TheCD spectra show the characteristic maximum at ˜227 nm seen for alltriple helices, and cooperative transitions were observed during allthree thermal melts. FIG. 8C is a plot of T_(m) values for a(mep-Pro-Gly)₇ triple helix versus TMAO concentration. Linear regressionand extrapolation to 0 M TMAO predicts a T_(m) value of 17.7° C. for a(mep-Pro-Gly)₇ triple helix, which is similar to the T_(m) value of 13°C. determined by direct measurement (FIG. 1B and Table 1).

Example 21 Sedimentation Equilibrium Experiments on (mep-Pro-Gly)₇,(Pro-Mep-Gly)₇, and (mep-Mep-Gly)₇

Sedimentation equilibrium experiments were performed with a Beckman XL-AAnalytical Ultracentrifuge at the University of Wisconsin BiophysicsInstrumentation Facility. Samples were diluted to approximately 0.1 mMin 50 mM potassium phosphate buffer (pH 3) and equilibrated at <4° C.for >24 h. Equilibrium data were collected at multiple speeds at both 4and 37° C. Gradients were monitored at 230 nm. Solvent densities of1.00494 and 0.99800 g/mL at 4 and 37° C., respectively, were measured byan Anton Paar DMA5000 density meter. Partial specific volumes ( ν) for(mep-Pro-Gly)₇, (Pro-Mep-Gly)₇ and (mep-Mep-Gly)₇ were calculated basedon amino acid content and corrected for the monomer molecular weightsdetermined by sedimentation equilibrium experiments at 37° C. A ν valueof 0.781 cm³/g was used for (mep-Pro-Gly)₇ and (Pro-Mep-Gly)₇ and a νvalue of 0.770 cm³/g was used for (mep-Mep-Gly)₇. Data were analyzedwith programs written for Igor Pro (Wavemetrics) by Dr. Darrell R.McCaslin (University of Wisconsin Biophysics Instrumentation Facility).

A log plot of absorbance versus the square of the distance from thecenter of rotation is shown in FIG. 9. The slope at any point isproportional to the weight-averaged molecular weight, provided that theextinction coefficients per unit mass of assembled and monomericpeptides are equivalent. Curvature in such plots demonstrates thepresence of multiple species.

Sedimentation equilibrium results at 37° C. are consistent with a singlemonomeric species for (mep-Pro-Gly)₇, (Pro-Mep-Gly)₇, and(mep-Mep-Gly)₇, as shown in FIG. 9. At 4° C., the dramatic change ingradient for (Pro-Mep-Gly)₇ and (mep-Mep-Gly)₇ is consistent with theassembly of these species into a triple helix. The fit shown at 4° C.for these two peptides (FIG. 9) is based on a mixture of monomer andtrimer. The data at 4° C. for (mep-Pro-Gly)₇ indicates some assembly forthis peptide at low temperature, but to a much lesser extent than isobserved for the other two peptides. The fit shown in FIG. 9 for(mep-Pro-Gly)₇ at 4° C. is for a mixture of monomer and trimer.

Example 22 Computations

The conformational preferences of 4-methylprolines were examined byhybrid density functional theory as implemented in Gaussian 98, RevisionA.9, M. J. Frisch, G. et al. Gaussian, Inc., Pittsburgh Pa., 1998.N-Acetyl-4-methylproline methyl esters were used as model compounds inthis study. Geometry optimizations and frequency calculations at theB3LYP/6−31+G* level of theory were performed on both the endo and exoconformers of Ac-mep-OMe and Ac-Mep-OMe, which were held in the trans(ω=1800) conformation. Frequency calculations of the optimizedstructures yielded no imaginary frequencies, indicating a truestationary point on the potential energy surface. Single-point energycalculations at the B3LYP/6−311+G(2d,p) level of theory were performedon the optimized structures. The resulting self-consistent field (SCF)energies were corrected by the zero-point vibrational energy (ZPVE)determined in the frequency calculations, and are listed in FIG. 7.

Example 23 Reciprocity of Steric and Stereoelectronic Effects in theCollagen Triple Helix

Density functional theory indicated that the pyrrolidine ring of(2S,4R)-4-methylproline (mep) has a strong preference (1.4 kcal/mol) forthe Cy-endo pucker and that of (2S,4S)-4-methylproline (Mep) has astrong preference (1.7 kcal/mol) for the Cy-exo pucker. Theseconformational preferences were observed in crystalline Ac-mep-NHMe andAc-Mep-NHMe by Flippen-Anderson et al., J. Am. Chem. Soc., 105:6609-6614(1983), and follow the trend observed in 4-tert-butylprolines byKoskinen et al., J. Org. Chem., 70:6447-6453 (2005). In the preferredconformations, the methyl group of mep and Mep adopts apseudo-equatorial conformation. A methyl group in this conformationshould protrude radially from a collagen triple helix and thus notinstill any deleterious steric interactions between the strands of thehelix. Accordingly, we synthesized mepOH and MepOH by the method of DelValle & Goodman, J. Org. Chem., 70:6447-6453 (2005) and incorporatedthese nonnatural amino acids into collagen strands to yield:(mep-Pro-Gly)₇, (Pro-Mep-Gly)₇, and (mep-Mep-Gly)₇. The incorporationinto collagen polymers was performed in the same manner reported in ourearlier work in this area, such as U.S. Pat. Nos. 5,973,112 and7,122,521, both of which are incorporated by reference herein in theirentirety. We incubated solutions of each strand at less than or about 4°C., and then used circular dichroism (CD) spectroscopy to detectformation of triple helices and assess their conformational stability.In a similar manner we also made (flp-Mep-Gly)₇ and (mep-Flp-Gly)₇.

(mep-Pro-Gly)₇, (Pro-Mep-Gly)₇, and (mep-Mep-Gly)₇ formed triple helicesat 4° C., as indicated by an ellipiticity maximum near 225 nm (FIG. 2A).The self-association of (Pro-Mep-Gly)₇, (mep-Mep-Gly)₇, and, to a lesserextent, (mep-Pro-Gly)₇ at 4° C. was confirmed by sedimentationequilibrium experiments. (mep-Pro-Gly)₇, (Pro-Mep-Gly)₇, and(mep-Mep-Gly)₇ triple helices had T_(m) values of 13, 29, and 36° C.,respectively, which are much greater than that of (Pro-Pro-Gly)₇. Theeffect of 4-Methylproline and 4-Fluoroproline diastereomers on theconformational stability of the collagen triple helix is shown in Table1 below. Thus, we conclude that steric effects can indeed stabilize thecollagen triple helix. Notably, CD experiments in solutions containingthe osmolyte trimethylamine-N-oxide confirm that triple helices of(mep-Pro-Gly)₇ have a T_(m) value near 13° C., but the low molarellipticity at 227 nm (FIG. 2A) and the results of sedimentationequilibrium experiments suggest that (mep-Pro-Gly)₇ is only partiallyassembled at 4° C. TABLE 1 T_(m) Tripeptide (±1° C.) Ref (mep-Flp-Gly)₇55 Described herein (flp-Mep-Gly)₇ 51 Described herein (Pro-Flp-Gly)₇ 45J. Am. Chem. Soc. 123, 777-778 (2003) (mep-Mep-Gly)₇ 36 Described herein(Pro-Hyp-Gly)₇ 36 J. Am. Chem. Soc. 123, 777-778 (2003) (flp-Pro-Gly)₇33 J. Am. Chem. Soc. 125, 9262-9263 (2003) (Pro-Mep-Gly)₇ 29 Describedherein (mep-Pro-Gly)₇ 13 Described herein (flp-Flp-Gly)₇  8^(a) J. Am.Chem. Soc. 127, 15923-15932 (2005) (Pro-Pro-Gly)₇ −6^(a) J. Am. Chem.Soc. 127, 15923-15932 (2005)(^(a)Based on the extrapolation of data from solutions containingtrimethylamine N-oxide.)

From the data in Table 1, it can be seen that Mep in the Yaa positionconferred more stability to a triple helix than does mep in the Xaaposition. Likewise, (2S,4R)-4-fluoroproline (Flp) in the Yaa positionincreased triple-helical propensity more than did(2S,4S)-4-fluoroproline (flp) in the Xaa position. We suspected thatthis dichotomy could arise from the effect of the steric andstereoelectronic effects on the peptide bond itself.

To determine the effect of a 4-methyl group on the value ofK_(trans/cis), we synthesized [¹³CH₃]Ac-mep-OMe and [¹³CH₃]Ac-Mep-OMeand evaluated K_(trans/cis) with ¹³C NMR spectroscopy. The trans:cisratio was twofold greater for Ac-Mep-OMe (K_(trans/cis)=7.4) than forAc-mep-OMe (K_(trans/cis)=3.6). These data provide an explanation fortriple helices formed by (Pro-Mep-Gly)₇ being more stable than thoseformed by (mep-Pro-Gly)₇. Apparently, a balance exists betweenpreorganization of the proper ring pucker and stabilization of a transpeptide bond.

These findings have numerous implications. Only recently werestereoelectronic effects found to contribute to the conformationalstability of a protein. Herein, steric effects are shown to reiteratethose same stereoelectronic effects. The stability of a non-natural(mep-Mep-Gly)₇ triple helix is indistinguishable from that of the“natural” (Pro-Hyp-Gly)₇ triple helix (Table 1), indicating thatside-chain heteroatoms (and hence side-chain solvation) are notnecessary for the formation of a stable triple helix. Thestereoelectronic effects induced by heteroatoms are not additive incollagen. A (flp-Flp-Gly)₇ triple helix is less stable than is a(flp-Pro-Gly)₇ or (Pro-Flp-Gly)₇ triple helix (Table 1), presumablybecause of an unfavorable steric interaction between fluoro groups onadjacent strands. In contrast, the steric effects are additive, as a(mep-Mep-Gly)₇ triple helix is more stable than is a (mep-Pro-Gly)₇ or(Pro-Mep-Gly)₇ triple helix (Table 1). The methyl groups of mep and Mepin synthetic collagen can likely be elaborated to larger functionalitieswithout undesirable encumbrance. We imagine the creation of a new classof hyperstable collagen mimetics by the judicious integration ofstereoelectronic and steric effects. The application of these venerableprinciples coupled with recent advances in the self-assembly of collagenfragments provides the means to create sturdy synthetic collagens forapplications in biomedicine and biotechnology.

The final evidence of the validity of this approach is the synthesis,performed using the procedures described above, of (flp-Mep-Gly)₇ and(mep-Flp-Gly)₇. These variants were found to have a T_(m) for(flp-Mep-Gly)₇ of 51° C. and a T_(m) for (mep-Flp-Gly)₇ of 55° C., bothof which represent a new plateau for collagen mimic stability.

Example 24 Synthesis ofN-tert-butyloxycarbonyl-(2S,4R)-4-thioacetyl-proline benzyl ester (16)

Diisopropyl azodicarboxylate (6.29 g, 31.1 mmol) was added dropwise at0° C. to a solution of triphenyl phosphine (8.16 g, 31.1 mmol) inanhydrous THF (87 mL). The solution was stirred at 0° C. 30 min. Asolution of thiolacetic acid (2.36 g, 31.1 mmol) andN-tert-butyloxycarbonyl-(2S,4S)-hydroxyproline benzyl ester (5.00 g,15.6 mmol) in anhydrous THF (25 mL) was added dropwise via cannula tothe first solution. The reaction mixture was stirred at 0° C. for 1 h,at rt for an additional 1 h and then concentrated under reducedpressure. Flash chromatography (first column: 45% v/v EtOAc in hexane toelute the desired product 16 in a mixture of other byproducts of similarpolarity, second column: 6% v/v EtOAc in hexane removed some of thebyproducts, and a third column: gradient: 6% v/v EtOAc in hexane to 50%EtOAc in hexane) afforded 16 (3.53 g. 9.6 mmol, 61.5%) as a colorlessoil. ¹H NMR δ: 1.34 and 1.45 (s, 9H), 2.14-2.46 (m, 2H), 2.32 (s, 3H),3.32 and 3.44 (dd, J=6.4, 10.8, 1H), 3.93 (m, 1H), 4.04 (m, 1H), 4.35and 4.46 (dd, J=5.1, 8.0, 1H), 5.08-5.29 (m, 2H), 7.35 (m, 5H); ¹³C NMRδ: 28.3, 28.5, 30.7, 35.8, 37.1, 39.5, 39.8, 51.6, 52.1, 58.4, 58.7,67.1, 80.7, 128.2, 128.4, 128.5, 128.6, 128.7, 128.8, 135.5, 135.7,153.5, 154.1, 172.0, 172.3, 194.8, 194.9; HRMS-ESI (m/z): [M+Na]⁺ calcdC₁₉H₂₅NO₅SNa, 402.1351; found, 402.1333.

Example 25 Synthesis of N-tert-butyloxycarbonyl-(2S,4R)-4-thioproline(17)

Compound 16 (1.62 g, 4.3 mmol) was dissolved in MeOH (27 mL). 2 Naqueous lithium hydroxide (13.5 mL) was added and the reaction mixturewas stirred at rt for 45 min. The reaction mixture was diluted withsaturated aqueous NaHCO₃ (100 μL) and the MeOH was removed by rotaryevaporation. The remaining aqueous solution was washed with ether (3×150mL), acidified with 2 N aqueous HCl to pH=1, and the acidic aqueoussolution was extracted with ether (3×225 mL). The ethereal solution wasdried with MgSO₄ and concentrated to afford 17 (0.67 g, 63%) as a whitesolid. ¹H NMR δ: 1.40-1.68 (2 s, 9H), 1.73 (m, 1H), 2.11-2.79 (m, 2H),3.41-3.98 (m, 4H), 8.8-9.2 (bs, 1H).

Example 26 Synthesis ofN-tert-butyloxycarbonyl-(2S,4R)-4-tritylthio-proline (18)

Compound 17 (0.66 g, 2.7 mmol) was dissolved in DMF (20 mL) andtrimethylamine (0.60 g, 5.9 mmol) was added followed by trityl chloride(0.90 g, 3.2 mmol). The reaction mixture was stirred under Ar(g) for 3h. The DMF was then removed by rotary evaporation under high vacuum. Theresidue was taken up in 10% w/v aqueous citric acid which was thenextracted with ether (100 mL) and ethyl acetate (2×125 mL). The combinedorganic layers were dried with MgSO₄ and concentrated. Flashchromatography over silica gel in 1:1 v/v EtOAc:hexane afforded 18 (1.53g, 57%) as a white solid.

Example 27 Synthesis ofN-9-Fluorenylmethoxycarbonyl-(2S,4R)-4-tritylthio-proline (19)

Compound 18 (0.75 g, 1.5 mmol) was dissolved in anhydrousdichloromethane (10 mL) under Ar(g) and the solution was cooled to −15°C. Trifluoroacetic acid (5 mL) was added and the resulting solution wasstirred for 4 h, slowly warming to rt over that time period. Afterconcentration, the residue was dissolved in 10% w/v aqueous NaHCO₃ (18mL) and a solution of Fmoc-OSu (0.57 g, 1.7 mmol) in 1,4-dioxane (25 mL)was added. The resulting solution was stirred for 23 h. The 1,4-dioxanewas removed by rotary evaporation and the basic aqueous slurry wasacidified with 2 N HCl to pH=1, extracted with EtOAc (3×150 mL), driedwith MgSO₄ and concentrated. Flash chromatography over silica gel in 1:3v/v EtOAc:hexane containing 0.1% formic acid afforded 19 (0.35 g, 35%)as a white solid. ¹H NMR δ: 1.18-1.39 (m, 2H), 1.82-2.14 (m, 1H),2.81-3.58 (m, 2H), 4.05-4.45 (m, 3H), 7.12-7.79 (m, 23H).

All of the documents cited herein are incorporated by reference here intheir entirety).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused.

1. A collagen mimic comprising a tripeptide having the formula:(Xaa-Yaa-Gly)n, where Xaa is a proline or proline derivative, where Yaais a proline or proline derivative, wherein the proline derivative is a4-substituted proline residue including any bulky and non-electronwithdrawing or electron donating substituent, and wherein thesubstituent is capable of stabilizing through steric hinderance effectsthe collagen mimic relative to a native collagen, and n is a positiveinteger.
 2. The collagen mimic of claim 1, wherein Xaa is a(2S,4R)-4-alkyl proline or a (2S,4R)-4-thioproline, and wherein anelectronegative atom including N, O, F, Cl, or Br is not installeddirectly on C4 of the proline ring.
 3. The collagen mimic of claim 2,wherein the (2S,4R)-4-alkyl proline is selected from the groupconsisting of 4-methylproline, 4-ethylproline, 4-propylproline,4-isopropylproline, or a longer alkyl proline.
 4. The collagen mimic ofclaim 1, wherein Yaa is a (2S,4S)-4-alkyl proline or a(2S,4S)-4-thioproline, and wherein an electronegative atom including N,O, F, Cl, or Br is not installed directly on C4 of the proline ring. 5.The collagen mimic of claim 4, wherein the (2S,4S)-4-alkyl proline isselected from the group consisting of 4-methylproline, 4-ethylproline,4-propylproline, 4-isopropylproline, or a longer alkyl proline.
 6. Thecollagen mimic of claim 1, wherein the tripeptide is selected from thegroup consisting of (Pro-Mep-Gly)n, (mep-Pro-Gly)n, (mep-Mep-Gly)n,(flp-Mep-Gly)n, (mep-Flp-Gly)n, (thp-Thp-Gly)n, (thp-Mep-Gly)n,(mep-Thp-Gly)n, (Pro-Thp-Gly)n, (thp-Pro-Gly)n, (thp-Hyp-Gly)n,(flp-Thp-Gly)n, and (thp-Flp-Gly)n, where n is a positive integer. 7.The collagen mimic of claim 6, wherein n is at least
 3. 8. A collagenmimic comprising a tripeptide having the formula:(Xaa-Yaa-Gly)n, where Xaa is a proline or proline derivative, where Yaais a proline or proline derivative, and wherein Xaa is(2S,4R)-4-methylproline or Yaa is (2S,4S)-4-methylproline, and n is apositive integer.
 9. The collagen mimic of claim 8, wherein n is atleast
 3. 10. The collagen mimic of claim 8, wherein the tripeptide ispresent in at least one out of every three triplex repeats.
 11. Acomposition of matter comprising a triple helix of collagen mimicmolecules in which each of the molecules in the helix comprisestripeptides of the formula:(flp-Yaa-Gly)n, where Yaa is a (2S,4S)-4-alkylproline or a(2S,4S)-4-thioproline, where flp is (2S,4S)-4-fluoroproline, and n is apositive integer.
 12. A composition of matter as claimed in claim 11wherein n is at least
 3. 13. A composition of matter comprising a triplehelix of collagen mimic molecules in which each of the molecules in thehelix comprises tripeptides of the formula:(Xaa-Flp-Gly)n, where Xaa is selected from the group consisting of(2S,4R)-4-alkylproline or a (2S,4R)-4-thioproline, where Flp is(2S,4R)-4-fluoroproline, and n is a positive integer.
 14. The peptide ofclaim 13, wherein n is at least
 3. 15. A collagen mimic comprising atripeptide having the formula:(Xaa-Yaa-Gly)n, where Xaa is a proline or proline derivatives, where Yaais a proline or proline derivatives, and wherein Xaa is(2S,4R)-4-thioproline or Yaa is (2S,4S)-4-thioproline, and n is apositive integer.
 16. The collagen mimic of claim 15, wherein n is atleast
 3. 17. The collagen mimic of claim 15, wherein the tripeptide ispresent in at least one out of every three triplex repeats.